scholarly journals Mechanistic Studies of the Yeast Polyamine Oxidase Fms1: Kinetic Mechanism, Substrate Specificity, and pH Dependence

Biochemistry ◽  
2010 ◽  
Vol 49 (49) ◽  
pp. 10440-10448 ◽  
Author(s):  
Mariya S. Adachi ◽  
Jason M. Torres ◽  
Paul F. Fitzpatrick
Keyword(s):  
Substrate Specificity ◽  
Ph Dependence ◽  
Kinetic Mechanism ◽  
Polyamine Oxidase ◽  
Mechanistic Studies

scholarly journals pH Dependence of a Mammalian Polyamine Oxidase: Insights into Substrate Specificity and the Role of Lysine 315†

Biochemistry ◽  
2009 ◽  
Vol 48 (7) ◽  
pp. 1508-1516 ◽  
Author(s):  
Michelle Henderson Pozzi ◽  
Vijay Gawandi ◽  
Paul F. Fitzpatrick
Keyword(s):  
Substrate Specificity ◽  
Ph Dependence ◽  
Polyamine Oxidase

scholarly journals Substrate specificity of sheep liver sorbitol dehydrogenase

1998 ◽  
Vol 330 (1) ◽  
pp. 479-487 ◽  
Author(s):  
I. Rune LINDSTAD ◽  
Peter KÖLL ◽  
John S. McKINLEY-McKEE
Keyword(s):  
Substrate Specificity ◽  
Ternary Complex ◽  
Kinetic Mechanism ◽  
Sorbitol Dehydrogenase ◽  
Hydroxyl Group ◽  
Enzyme Active Site ◽  
Sheep Liver ◽  
Rate Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo > d-ribo > L-xylo > d-lyxo ≈ l-arabino > D-arabino > l-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH >-CH2NH2 >-CH2OCH3 ≈-CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

Nitroalkanes as Reductive Substrates for Flavoprotein Oxidases

1972 ◽  
Vol 27 (9) ◽  
pp. 1052-1053 ◽  
Author(s):  
David J. T. Porter ◽  
Judith G. Voet ◽  
Harold J. Bright
Keyword(s):  
Hydrogen Peroxide ◽  
Amino Acid ◽  
Glucose Oxidase ◽  
Ph Dependence ◽  
Kinetic Mechanism ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Kinetics Of ◽  
Oxidase Reaction ◽  
Detailed Kinetic Mechanism

Nitroalkanes have been found to be general reductive substrates for D-amino acid oxidase, glucose oxidase and L-amino acid oxidase. These enzymes show different specificities for the structure of the nitroalkane substrate.The stoichiometry of the D-amino acid oxidase reaction is straightforward, consisting of the production of one mole each of aldehyde, nitrite and hydrogen peroxide for each mole of nitroalkane and oxygen consumed. The stoichiometry of the glucose oxidase reaction is more complex in that less than one mole of hydrogen peroxide and nitrite is produced and nitrate and traces of 1-dinitroalkane are formed.The kinetics of nitroalkane oxidation show that the nitroalkane anion is much more reactive in reducing the flavin than is the neutral substrate. The pH dependence of flavin reduction strongly suggests that proton abstraction is a necessary event in catalysis. A detailed kinetic mechanism is presented for the oxidation of nitroethane by glucose.It has been possible to trap a form of modified flavin in the reaction of D-amino acid oxidase with nitromethane from which oxidized FAD can be regenerated in aqueous solution in the presence of oxygen.

Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune

2001 ◽  
Vol 356 (3) ◽  
pp. 757-767 ◽  
Author(s):  
Christian EIS ◽  
Mark WATKINS ◽  
Thomas PROHASKA ◽  
Bernd NIDETZKY
Keyword(s):  
Inductively Coupled Plasma ◽  
Metal Ion ◽  
Ph Dependence ◽  
Schizophyllum Commune ◽  
Structural Similarity ◽  
Kinetic Mechanism ◽  
Inductively Coupled ◽  
Peptide Mass ◽  
Dead End ◽  
Trehalose Phosphorylase

Initial-velocity measurements for the phospholysis and synthesis of α,α-trehalose catalysed by trehalose phosphorylase from Schizophyllum commune and product and dead-end inhibitor studies show that this enzyme has an ordered Bi Bi kinetic mechanism, in which phosphate binds before α,α-trehalose, and α-d-glucose is released before α-d-glucose 1-phosphate. The free-energy profile for the enzymic reaction at physiological reactant concentrations displays its largest barriers for steps involved in reverse glucosyl transfer to d-glucose, and reveals the direction of phospholysis to be favoured thermodynamically. The pH dependence of kinetic parameters for all substrates and the dissociation constant of d-glucal, a competitive dead-end inhibitor against d-glucose (Ki = 0.3mM at pH6.6 and 30°C), were determined. Maximum velocities and catalytic efficiencies for the forward and reverse reactions decrease at high and low pH, giving apparent pK values of 7.2–7.8 and 5.5–6.0 for two groups whose correct protonation state is required for catalysis. The pH dependences of kcat/K are interpreted in terms of monoanionic phosphate and α-d-glucose 1-phosphate being the substrates, and of the pK value seen at high pH corresponding to the phosphate group in solution or bound to the enzyme. The Ki value for the inhibitor decreases outside the optimum pH range for catalysis, indicating that binding of d-glucal is tighter with incorrectly ionized forms of the complex between the enzyme and α-d-glucose 1-phosphate. Each molecule of trehalose phosphorylase contains one Mg2+ that is non-dissociable in the presence of metal chelators. Measurements of the 26Mg2+/24Mg2+ ratio in the solvent and on the enzyme by using inductively coupled plasma MS show that exchange of metal ion between protein and solution does not occur at measurable rates. Tryptic peptide mass mapping reveals close structural similarity between trehalose phosphorylases from basidiomycete fungi.

Mechanistic studies of the flavoprotein tryptophan 2-monooxygenase. 1. Kinetic mechanism

Biochemistry ◽  
1995 ◽  
Vol 34 (11) ◽  
pp. 3710-3715 ◽  
Author(s):  
John J. Emanuele ◽  
Paul F. Fitzpatrick
Keyword(s):  
Kinetic Mechanism ◽  
Mechanistic Studies

scholarly journals Substrate Specificity and Kinetic Mechanism of Mammalian G9a Histone H3 Methyltransferase

2004 ◽  
Vol 279 (51) ◽  
pp. 53248-53258 ◽  
Author(s):  
Debasis Patnaik ◽  
Hang Gyeong Chin ◽  
Pierre-Olivier Estève ◽  
Jack Benner ◽  
Steven E. Jacobsen ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Histone H3 ◽  
Kinetic Mechanism
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